Folate is essential for the human and animal diet and deficiency can result in a range of diseases and disorders, such as neural tube defects in newborns. Many plants, fungi and bacteria synthesize folate, including lactic bacteria (LAB), whereby folate concentrations in fermented dairy products are higher than in non-fermented dairy product. The annotated genome sequence of Lactococcus lactis subsp. lactis IL1403 shows the genes of the folate biosynthetic cluster. The folate genes from other gene clusters have also been identified, such as those of Lactococcus lactis MG1363, and are used to genetically engineer bacteria having increased folate production, for example through overexpression of the folKE gene (Sybesma et al., 2003, Applied and Environm. Microb. Vol. 69(6), pp 3069-3076). Controlled overexpression of folKE resulted in a 3-fold increase of total folate production and a 10-fold increase of extracellular folate production.
Not all LAB are able to produce folate. For example, Sybesma et al. (2003, Applied and Environm. Microbiology Vol. 69(8) pp 4542-4548) found that while Lactococcus lactis, Streptococcus thermophilus and Leuconostoc species were able to produce folate, most Lactobacillus species tested were not, with the exception of L. plantarum. Total (intracellular and extracellular) deconjugated folate levels varied widely and maximum levels were 291 μg/L for strain L. lactis ssp. lactis NZ9010 (grown aerobically on M17 culture medium; strain is defective in lactate dehydrogenase), while L. plantarum WCFS 1 for example only produced 45 μg/L on MRS medium. Also the proportion excreted into the medium varied.
Sybesma et al. (supra) also studied the effects of culture conditions such as pH, p-ABA and hemin on folate production and distribution in two commonly used LAB, L. lactis MG1363 and S. thermophilus NIZO strain B119. Culture media included M17 medium, Chemically Defined Medium (CDM) and MRS medium. In continuous culture an increase in pH from 5.5 to 7.5 resulted in an increased folate production in these two strains by a factor 2-3, reaching 534 μs/L for S. thermophilus grown on M17 medium and 107 μg/L for L. lactis grown on CDM. For L. lactis, the addition of p-ABA in concentrations ranging from 1-100 μM also increased folate production, while amounts of p-ABA above 100 μM had no effect. Interestingly, the addition of growth inhibiting substances also resulted in a folate increase.
As already mentioned, Lactobacillus species produce no or very low levels of folate. Lactobacillus species are important in the production of fermented products and an increase in folate production by those species would be highly desirable. Lactobacillus species are for example used industrially for the production of yogurt, cheese, sauerkraut, pickles, and other fermented foods, as well as animal feeds, such as silage. In addition, the use of natural (food grade), rather than synthetic, fermentation media has the advantage that the natural media can be used as such for food-, feed- or food supplement production, eliminating the need for purifying the folate from the culture. These objectives are met by the present invention.
WO02/097063 describes the production of bioavailable folic acid by recombinant microorganisms (genetically modified bacteria). The recombinant bacteria are grown on synthetic media.
WO2006/013588 describes folic acid producing probiotic Bifidobacterium strains and their use. These may be used in formulations together with other probiotic LAB.
WO2006/093408 relates to mutant bacteria which are resistant to methotrexate and overproduce folate. The bacteria are identified by using methotrexate as a selection agent. There is no indication that natural fermentation media may increase folate production.